Project Summary Membrane fusion between gametes (e.g. sperm & egg) during fertilization is a crucial step in eukaryotic life cycles. Unfortunately, not much is known about the molecules regulating this vital reproductive process. One protein that is known to be important is HAP2, a conserved, male-gamete- specific factor necessary for fertilization in a wide range of eukaryotic species, including many important human and animal pathogens (e.g. Plasmodium sp. causing malaria, Trypanosoma, Toxoplasma, Eimeria, etc.). Exciting recent work has discovered that HAP2 is structurally homologous to viral class II fusion proteins. These viral (e.g. Dengue and Zika) class II proteins mediate the merger of virus and host cell membranes during infection and are dependent upon oligomeric and conformational changes in response to low pH for their fusogenic activity. The proposed studies will test the model that, before fusion, HAP2 organizes itself on the gamete membrane in a similar way to certain viral class II proteins, which in turn, helps regulate HAP2's function in gamete cell fusion during sex. The experimentally- amenable sexual life cycle of the unicellular micro-alga, Chlamydomonas reinhardtii, will be used as a model system. Genetically-tractable Chlamydomonas cells readily form plus and minus gametes in the laboratory and well-established methods exist for quantifying the distinct steps in the gamete membrane fusion reaction. Chlamydomonas is also the only organism in which a HAP2 protein structure has been published and where a receptor-binding protein which interacts with HAP2 has been identified (MAR1, preliminary data). Furthermore, using strategically designed mutations of Chlamydomonas HAP2, along with biochemical crosslinking methods, we have detected oligomeric forms of this fusogen which offer tantalizing clues concerning its multimeric contacts on the membrane before fusion. The long-term objectives of this proposal are to enhance our fundamental understanding of the mechanism of membrane fusion at fertilization and to identify additional therapeutic targets on HAP2 which could lead to better vaccines or prevention strategies for many harmful parasitic diseases.